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Polypeptide cross-linked protein molecularly imprinted polymer and its preparation method and application

A molecular imprinting and polymer technology, applied in chemical instruments and methods, peptides, specific peptides, etc., can solve problems such as poor imprinting effect and difficult template elution

Active Publication Date: 2020-05-19
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] For western blotting, the problem of difficult template elution and poor blotting seems to be an inherent problem, and the two problems are intertwined, and it is difficult to solve them at the same time
People have not been able to find an effective solution after a long period of research, making the prospect of western blotting cast a shadow (Culver, H.R.; Peppas, N.A., Chemistry of Materials, 2017, 29(14), 5753-5761)

Method used

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  • Polypeptide cross-linked protein molecularly imprinted polymer and its preparation method and application
  • Polypeptide cross-linked protein molecularly imprinted polymer and its preparation method and application
  • Polypeptide cross-linked protein molecularly imprinted polymer and its preparation method and application

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preparation example Construction

[0029] The invention provides a method for preparing a polypeptide crosslinked protein molecularly imprinted polymer, comprising the following steps: 1) mixing and dissolving a main monomer, a functional monomer, a polypeptide crosslinking agent and a template protein in a water-soluble solvent to obtain a mixed solution; 2) adding an initiator to the mixed solution described in step 1), and polymerizing under the condition that the polypeptide cross-linking agent exists in a helix conformation to obtain a polymer; 3) under the condition that the polypeptide cross-linking agent exists in a coil conformation, Perform template elution on the polymer described in step 2) to obtain a protein molecularly imprinted polymer cross-linked by the polypeptide; the polypeptide cross-linking agent has double bonds capable of polymerizing at both ends of the amino acid sequence, and can generate helix-coil Conformational switched peptides.

[0030]In the present invention, the polypeptide c...

Embodiment 1

[0042] Synthesis of imprinted polymers:

[0043] 200 mg of N-isopropylacrylamide, 5 mg of acrylamide, 4 μL of methacrylic acid, 115 mg of polypeptide cross-linker and 100 mg of template protein lysozyme were dissolved in 2 mL of 20 mM pH5.5 phosphate buffer solution. After thorough mixing, 50 μL of 10% APS and 5 μL TEMED were added to initiate polymerization, and reacted at 37° C. for 24 hours to obtain the imprinted polymer MIP. The non-imprinted polymer NIP was prepared in the same way as the imprinted polymer, except that the template protein lysozyme was not added. The structure of the polypeptide cross-linking agent is shown in formula I:

[0044]

[0045] Elution of template protein:

[0046] Protein elution was carried out at 37° C. with 20 mM pH7.4 phosphate buffer solution (containing 0.154 M NaCl to simulate physiological ionic strength). Measure the absorbance value of the eluate, use a UV spectrophotometer to draw a standard curve of absorbance-concentration ...

Embodiment 2

[0060] Synthesis of imprinted polymers:

[0061] Dissolve 200 mg N-isopropylacrylamide, 2.5 mg acrylamide, 8 μL dimethylaminoethyl methacrylate, 115 mg polypeptide cross-linker and 100 mg template protein bovine serum albumin (BSA) in 2 mL 20 mM pH5.5 phosphate buffer solution. After mixing well, add initiator 50 μL 10% APS and 5 μL TEMED, react at 37° C. for 24 h. Obtain the imprinted polymer MIP. The non-imprinted polymer NIP was prepared in the same way as the imprinted polymer, except that the template protein BSA was not added. The structure of the polypeptide cross-linking agent is the same as in Example 1.

[0062] Elution of template protein:

[0063] Protein was eluted with 20mM pH7.4 phosphate buffer solution (containing 0.154M NaCl to simulate physiological ionic strength) at 37°C, and the four elution rates were 95.24%, 2.05%, 0.24%, and 0%, respectively. The total elution rate was 97.51%. Finally, the imprinted polymer was washed with deionized water to remo...

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Abstract

The invention discloses a polypeptide-crosslinked protein molecularly imprinted polymer and a preparation method and application thereof, belonging to the technical field of biological separation engineering. The method comprises the following steps: 1) combining a main monomer, a functional monomer, a polypeptide cross-linking agent and a The template protein is mixed and dissolved in a water-soluble solvent to obtain a mixed solution; 2) The initiator is added to the mixed solution, and the polymerization is carried out under the condition that the polypeptide cross-linking agent exists in the helix conformation; 3) The polypeptide cross-linking agent exists in the coil conformation. Under certain conditions, template elution is performed to obtain a protein molecularly imprinted polymer cross-linked by a polypeptide; the polypeptide cross-linking agent is a polypeptide having double bonds capable of polymerizing at both ends of the amino acid sequence and capable of undergoing helix-coil conformational transition. The polypeptide-crosslinked protein molecularly imprinted polymer disclosed in the present invention can not only completely remove the template protein under mild conditions, but also can greatly improve the imprinting effect of the protein molecularly imprinted polymer.

Description

technical field [0001] The invention belongs to the technical field of biological separation engineering, and in particular relates to a polypeptide cross-linked protein molecularly imprinted polymer and a preparation method and application thereof. Background technique [0002] Molecularly imprinted polymers are a class of artificially synthesized receptors inspired by the specific recognition of antigens by antibodies. Compared with natural receptors such as antibodies, molecularly imprinted polymers have the advantages of simple preparation, good stability, and low cost, and have important applications in many fields such as the environment and life. The general process of molecular imprinting is: (1) polymerization of monomers and cross-linking agents in the presence of template molecules, (2) template elution, leaving imprints in the polymer that are chemically and spatially complementary to the template molecules sites, (3) the imprinted polymer recognizes and selecti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08F289/00C08F220/54C08J9/26B01J20/26B01J20/30
CPCC07K17/08C08L89/00C08F220/54C08F290/065C08F220/56C08F220/06C08F222/385C08F220/34C08F220/60C08F2/04C08F2/44C08F289/00
Inventor 张拥军许荣关英
Owner NANKAI UNIV
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